Changes in cytosolic Ca2+ levels correspond to fluctuations of lactate levels in crosstalk of astrocyte-neuron cell lines

Neurons and astrocytes differentially express isoenzymes of lactate dehydrogenase (LDH). The metabolic consequences for the variations in mRNA expression of LDH isoenzyme subtypes in neurons and astrocytes control cerebral vasoregulation. Moreover, cellular signalling consequences for functional neurovascular control may also be dependent on LDH isoenzyme subtype profiles. Initial computer simulations revealed glutamate-induced calcium waves in connected astrocytes, and showed concomitant changes in the expression of nitric oxide synthase (NOS) and lactic acid metabolism. To validate these findings, the nature and extent of glutamate-dependent signalling crosstalk in murine cell lines were investigated through correlated lactate levels and calcium upregulation. Neuro2A and C8D1A cells were separately treated with timed supernatant extracts from each other and their LDH1 and LDH5 isoenzyme responses were recorded. Western blot analysis showed LDH1/LDH5 isoenzyme ratio in the astrocytes to be positively correlated with Neuro2A-derived lactate levels estimated by the amplitude of 1.33-ppm spectral peak in 1H-NMR, and LDH1/LDH5 isoenzyme ratio in neurons is negatively correlated with CSD1A-derived lactate levels. Significant modulations of the calcium-responsive protein pCamKII levels were also observed in both cell lines, particularly correlations between pCamKII and lactate in C8D1A cells, thus explaining the calcium dependence of the lactate response. Together, these observations indicate that lactate is a key indicator of the metabolic state of these cell types, and may be a determinant of release of vasoregulatory factors.     

Characterization of Trichoderma reesei endoglucanase ii expressed heterologously in Pichia pastoris for better biofinishing and biostoning

The endoglucanase II of Trichoderma reesei is considered the most effective enzyme for biofinishing cotton fabrics and biostoning denim garments. However, the commercially available preparation of endoglucanase II is usually mixed with other cellulase components, especially endoglucanase I, resulting in hydrolysis and weight loss of garments during biofinishing and biostoning. We thus isolated the endoglucanase II gene from T. reesei to express this in Pichia pastoris, under the control of a methanol-inducible AOX1 promoter, to avoid the presence of other cellulase components. A highly expressible Mut(+) transformant was selected and its expression in BMMH medium was found most suitable for the production of large amounts of the recombinant protein. Recombinant endoglucanase II was purified to electrophoretic homogeneity, and functionally characterized by activity staining. The specific activity of recombinant endoglucanase II was found to be 220.57 EU/mg of protein. Purified recombinant endoglucanase II was estimated to have a molecular mass of 52.8 kDa. The increase in molecular mass was likely due to hyperglycosylation. Hyperglycosylation of recombinant endoglucanase II secreted by P. pastoris did not change the temperature or pH optima as compared to the native protein, but did result in increased thermostability. Kinetic analysis showed that recombinant endoglucanase was most active against amorphous cellulose, such as carboxymethyl cellulose, for which it also had a high affinity.     

Heme Oxygenase-1 Promotes Survival of Renal Cancer Cells through Modulation of Apoptosis- and Autophagy-regulating Molecules

The cytoprotective enzyme heme oxygenase-1 (HO-1) is often overexpressed in different types of cancers and promotes cancer progression. We have recently shown that the Ras-Raf-ERK pathway induces HO-1 to promote survival of renal cancer cells. Here, we examined the possible mechanisms underlying HO-1-mediated cell survival. Considering the growing evidence about the significance of apoptosis and autophagy in cancer, we tried to investigate how HO-1 controls these events to regulate survival of cancer cells. Rapamycin (RAPA) and sorafenib, two commonly used drugs for renal cancer treatment, were found to induce HO-1 expression in renal cancer cells Caki-1 and 786-O; and the apoptotic effect of these drugs was markedly enhanced upon HO-1 knockdown. Overexpression of HO-1 protected the cells from RAPA- and sorafenib-induced apoptosis and also averted drug-mediated inhibition of cell proliferation. HO-1 induced the expression of anti-apoptotic Bcl-xL and decreased the expression of autophagic proteins Beclin-1 and LC3B-II; while knockdown of HO-1 down-regulated Bcl-xL and markedly increased LC3B-II. Moreover, HO-1 promoted the association of Beclin-1 with Bcl-xL and Rubicon, a novel negative regulator of autophagy. Drug-induced dissociation of Beclin-1 from Rubicon and the induction of autophagy were also inhibited by HO-1. Together, our data signify that HO-1 is up-regulated in renal cancer cells as a survival strategy against chemotherapeutic drugs and promotes growth of tumor cells by inhibiting both apoptosis and autophagy. Thus, application of chemotherapeutic drugs along with HO-1 inhibitor may elevate therapeutic efficiency by reducing the cytoprotective effects of HO-1 and by simultaneous induction of both apoptosis and autophagy.     

Combining mathematics and empirical data to predict emergence of RNA viruses that differ in reservoir use

RNA viruses may be particularly capable of contributing to the increasing biomedical problem of infectious disease emergence. Empirical studies and epidemiological models are informative for the understanding of evolutionary processes that promote pathogen emergence, but rarely are these approaches combined in the same study. Here, we used an epidemiology model containing observations of pathogen productivity in reservoirs, as a means to predict which pathogens should be most prone to emerge in a primary host such as humans. We employed as a model system a collection of vesicular stomatitis virus populations that had previously diverged in host-use strategy: specialists, directly selected generalists and indirectly selected (fortuitous) generalists. Using data from experiments where these viral strategists were challenged to grow on unencountered novel hosts in vitro, logistic growth models determined that the directly selected generalist viruses tended to grow best on model reservoirs. Furthermore, when we used the growth data to estimate average reproductive rate across secondary reservoirs, we showed that the combined approach could be used to estimate relative success of the differing virus strategists when encountering a primary host. Our study suggests that synergistic approaches combining epidemiological modelling with empirical data from experimental evolution may be useful for developing efforts to predict which types of pathogens pose the greatest probability of emerging in the future.     

Proteome differences associated with fat accumulation in bovine subcutaneous adipose tissues

Background  

The fat components of red meat products have been of interest to researchers due to the health aspects of excess fat consumption by humans. We hypothesized that differences in protein expression have an impact on adipose tissue formation during beef cattle development and growth. Therefore, in this study we evaluated the differences in the discernable proteome of subcutaneous adipose tissues of 35 beef crossbred steers [Charolais × Red Angus (CHAR) (n = 13) and Hereford × Angus (HEAN) (n = 22)] with different back fat (BF) thicknesses. The goal was to identify specific protein markers that could be associated with adipose tissue formation in beef cows.     

Structure based virtual screening of GSK-3β: Importance of protein flexibility and induced fit

GSK-3β, one of the vital enzymes responsible for various phosphorylation catalysis. Induced fit mechanism and the presence of conserved water molecule(s) in the active site poses complexity during the process of virtual screening. The present investigation reveals the practical strategy to handle the induced fit mechanism of GSK-3β though flexible docking protocol. This protocol provides an enrichment of 70% in top 1% of the dataset with a rank correlation of >0.9 and found better in comparison to earlier reported protocols.     

Diguanylate cyclase NicD‐based signalling mechanism of nutrient‐induced dispersion by Pseudomonas aeruginosa

Dispersion enables the transition from the biofilm to the planktonic growth state in response to various cues. While several Pseudomonas aeruginosa proteins, including BdlA and the c‐di‐GMP phosphodiesterases DipA, RbdA, and NbdA, have been shown to be required for dispersion to occur, little is known about dispersion cue sensing and the signalling translating these cues into the modulation c‐di‐GMP levels to enable dispersion. Using glutamate‐induced dispersion as a model, we report that dispersion‐inducing nutrient cues are sensed via an outside‐in signalling mechanism by the diguanylate cyclase NicD belonging to a family of seven transmembrane (7TM) receptors. NicD directly interacts with BdlA and the phosphodiesterase DipA, with NicD, BdlA, and DipA being part of the same pathway required for dispersion. Glutamate sensing by NicD results in NicD dephosphorylation and increased cyclase activity. Active NicD contributes to the non‐processive proteolysis and activation of BdlA via phosphorylation and temporarily elevated c‐di‐GMP levels. BdlA, in turn, activates DipA, resulting in the overall reduction of c‐di‐GMP levels. Our results provide a basis for understanding the signalling mechanism based on NicD to induce biofilm dispersion that may be applicable to various biofilm‐forming species and may have implications for the control of biofilm‐related infections.     

The extinct,giant giraffid Sivatherium giganteum: skeletal reconstruction and body mass estimation

Sivatherium giganteum is an extinct giraffid from the Plio–Pleistocene boundary of the Himalayan foothills. To date, there has been no rigorous skeletal reconstruction of this unusual mammal. Historical and contemporary accounts anecdotally state that Sivatherium rivalled the African elephant in terms of its body mass, but this statement has never been tested. Here, we present a three-dimensional composite skeletal reconstruction and calculate a representative body mass estimate for this species using a volumetric method. We find that the estimated adult body mass of 1246 kg (857—1812 kg range) does not approach that of an African elephant, but confirms that Sivatherium was certainly a large giraffid, and may have been the largest ruminant mammal that has ever existed. We contrast this volumetric estimate with a bivariate scaling estimate derived from Sivatherium''s humeral circumference and find that there is a discrepancy between the two. The difference implies that the humeral circumference of Sivatherium is greater than expected for an animal of this size, and we speculate this may be linked to a cranial shift in centre of mass.